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International Urology and Nephrology
Published in: International Urology and Nephrology 8/2016

01-08-2016 | Urology - Original Paper

Carbachol-induced signaling through Thr696-phosphorylation of myosin phosphatase-targeting subunit 1 (MYPT1) in rat bladder smooth muscle cells

Authors: Benchun Liu, Yung-Chin Lee, Amjad Alwaal, Guifang Wang, Lia Banie, Ching-Shwun Lin, Guiting Lin, Tom F. Lue

Published in: International Urology and Nephrology | Issue 8/2016

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Abstract

Purpose

Lines of evidence suggest that Rho-associated protein kinase (ROCK)-mediated myosin phosphatase-targeting subunit 1 (MYPT1) phosphorylation plays a central role in smooth muscle contraction. However, the physiological significance of MYPT1 phosphorylation at Thr696 catalyzed by ROCK in bladder smooth muscle remains controversial. We attempt to directly observe the quantitative protein expression of Rho A/ROCK and phosphorylation of MYPT1 at Thr696 after carbachol administration in rat bladder smooth muscle cells (RBMSCs).

Materials and methods

Primary cultured smooth muscle cells were obtained from rat bladders. The effects of both concentration and time-course induced by the muscarinic agonist carbachol were investigated by assessing the expression of Rho A/ROCK and MYPT1 phosphorylation at Thr696 using Western blot.

Results

In the dose-course studies, carbachol showed significant increase in phosphorylation of MYPT1 at Thr696 (p-MYPT1) from concentrations of 15–100 μM based on Western blot results (p < 0.05, ANOVA test). In the time-course studies, treatment of cells with 15 μM of carbachol significantly enhanced the expression of p-MYPT1 from 3 to 15 h (p < 0.05, ANOVA test) and induced the expression of Rho A from 10 to 120 min (p < 0.05, ANOVA test).

Conclusions

Carbachol can induce the expression of ROCK pathway, leading to MYPT1 phosphorylation at Thr696 and thereby sustained RBSMCs contraction.
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Metadata
Title
Carbachol-induced signaling through Thr696-phosphorylation of myosin phosphatase-targeting subunit 1 (MYPT1) in rat bladder smooth muscle cells
Authors
Benchun Liu
Yung-Chin Lee
Amjad Alwaal
Guifang Wang
Lia Banie
Ching-Shwun Lin
Guiting Lin
Tom F. Lue
Publication date
01-08-2016
Publisher
Springer Netherlands
Published in
International Urology and Nephrology / Issue 8/2016
Print ISSN: 0301-1623
Electronic ISSN: 1573-2584
DOI
https://doi.org/10.1007/s11255-016-1303-2

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